NEUROPEPTIDE-Y STIMULATES LUTEINIZING-HORMONE-RELEASING HORMONE-RELEASE FROM SUPERFUSED HYPOTHALAMIC GT(1)-7 CELLS

The effects of neuropeptide Y (NPY) on LHRH release from an immortaliz ed cell line were investigated using a flow-through cell culture super fusion system. Immortalized hypothalamic GT(1)-7 cells were cultured f or 72 h and superfused for a total of 180 min. In initial experiments, discrete 5-min pulses of NPY (10(-12)-10(-5) M) were administered to the cells. A clear dose-dependent stimulatory effect of NPY on LHRH re lease from the cells was observed with a calculated 50% effectiveness concentration of 33 nM. The stimulatory effects of brief NPY exposure were rapid and robust, e.g. reaching and maintaining levels of 173% ov er baseline for 20 min at the 10(-7) dose. The lowest dose of NPY that showed a significant effect was 10(-10) M; maximal responses were obs erved at 10(-6) M and reached a plateau thereafter. Control pulses of Dulbecco's modified Eagle's medium (DMEM) and 10(-6) M substance P or arg-vasopressin were also presented to the cells to serve as controls for our pulse protocol, and these challenges produced no significant L HRH responses. The NPY receptor antagonists, PYX1 and PYX2, at 10(-8) M, completely blocked the observed NPY responses in these cells. To as sess the NPY receptor subtypes that mediate the NPY effects pharmacolo gically, GT(1)-7 cells were challenged with a Y-1 receptor agonist, (L eu(31)Pro(34))NPY, a Y-2 receptor agonist, NPY(13-36), or peptide YY, at doses 10(-12)-10(-5) M. All four peptides stimulated LHRH release f rom GT(1)-7 cells with a ranking ordered potency of NPY = peptide YY > Y-1 agonist = Y-2 agonist. To examine possible signal transduction me chanism(s) involved in mediating this effect, pertussis toxin, RpcAMPs (cyclic adenosine-3'5'-monophosphothioate Rp diastereomer), Ca2+-free DMEM and TMB-8 (3, 4, 5-trimethoxybenzoic acid 8-(diethylamino) octyl ester) were used to treat the cells before and during superfusion with NPY. Treatment with pertussis toxin, RpcAMPs, and Ca2+-free DMEM did not significantly alter NPY-stimulated LHRH release responses to 10(-7 ) M NPY. However, the addition of 100 mu M and 250 mu M TMB-8 to Ca2+- free DMEM almost completely blocked this NPY effect, as did 10 mu M ry anodine. Finally, the locus of action for this NPY effect was examined using tetrodotoxin to reduce action potential propagation in the GT(1 )-7 cells. Tetrodotoxin treatment blocked the LHRH response to NPY by more than 50% These results demonstrate for the first time that NPY di rectly stimulates LHRH release from superfused GT(1)-7 cells in a dose -dependent manner and that this effect seems to be specific to the NPY peptide. In addition, NPY may exert its effects on LHRH neurons predo minantly via receptors possessing Y-1 subtype pharmacological properti es. The effects of NPY also appear to be dependent upon the mobilizati on of intracellular calcium from IP3- and/or ryanodine-sensitive store s. Our findings, therefore, suggest that one mechanism by which NPY ma y stimulate LHRH release in vivo may be through a direct, Y-1-like rec eptor-mediated action on the LHRH neuron itself.